Lehrstuhl für Chemische Biologie der Nukleinsäuren
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Item Light‐activatable MBD‐readers of 5‐methylcytosine reveal domain‐dependent chromatin association kinetics in vivo(2024-01-02) Lin, Tzu-Chen; Engelhard, Lena; Söldner, Benedikt; Linser, Rasmus; Summerer, Daniel5-Methylcytosine (5mC) is the central epigenetic mark of mammalian DNA, and plays fundamental roles in chromatin regulation. 5mC is dynamically read and translated into regulatory outputs by methyl-CpG-binding domain (MBD) proteins. These multidomain readers recognize 5mC via an MBD domain, and undergo additional domain-dependent interactions with multiple additional chromatin components. However, studying this dynamic process is limited by a lack of methods to conditionally control the 5mC affinity of MBD readers in cells. Light-control of MBD association to chromatin by genetically encoding a photocaged serine at the MBD-DNA interface is reported. The authors study the association of MBD1 to mouse pericentromeres, dependent on its CxxC3 and transcriptional repressor domains (TRD) which interact with unmethylated CpG and heterochromatin-associated complexes, respectively. Both domains significantly modulate association kinetics, arguing for a model in which the CxxC3 delays methylation responses of MBD1 by holding it at unmethylated loci, whereas the TRD promotes responses by aiding heterochromatin association is studied. Their approach offers otherwise inaccessible kinetic insights into the domain-specific regulation of a central MBD reader, and sets the basis for further unravelling how the integration of MBDs into complex heterochromatin interaction networks control the kinetics of 5mC reading and translation into altered chromatin states.Item Programmable protein-DNA crosslinking for the direct capture and quantification of 5-formylcytosine(2019-06-04) Gieß, Mario; Munoz-Lopez, Alvaro; Buchmuller, Benjamin; Kubik, Grzegorz; Summerer, Daniel5-Formylcytosine (5fC) is an epigenetic nucleobase of mammalian genomes that occurs as intermediate of active DNA demethylation. 5fC uniquely interacts and reacts with key nuclear proteins, indicating functions in genome regulation. Transcription-activator-like effectors (TALEs) are repeat-based DNA binding proteins that can serve as probes for the direct, programmable recognition and analysis of epigenetic nucleobases. However, no TALE repeats for the selective recognition of 5fC are available, and the typically low genomic levels of 5fC represent a particular sensitivity challenge. We here advance TALEbased nucleobase targeting from recognition to covalent crosslinking. We report TALE repeats bearing the ketoneamino acid p-acetylphenylalanine (pAcF) that universally bind all mammalian cytosine nucleobases, but selectively form diaminooxy-linker-mediated dioxime crosslinks to 5fC. We identify repeat-linker combinations enabling single CpG resolution, and demonstrate the direct quantification of 5fC levels in a human genome background by covalent enrichment. This strategy provides a new avenue to expand the application scope of programmable probes with selectivity beyond A, G, T and C for epigenetic studies.Item Epigenetic chromatin modification by amber suppression technology(2018-02-22) Neumann, Heinz; Neumann-Staubitz, Petra; Witte, Anna; Summerer, DanielThe genetic incorporation of unnatural amino acids (UAAs) into proteins by amber suppression technology provides unique avenues to study protein structure, function and interactions both in vitro and in living cells and organisms. This approach has been particularly useful for studying mechanisms of epigenetic chromatin regulation, since these extensively involve dynamic changes in structure, complex formation and posttranslational modifications that are difficult to access by traditional approaches. Here, we review recent achievements in this field, emphasizing UAAs that help to unravel protein-protein interactions in cells by photo-crosslinking or that allow the biosynthesis of proteins with defined posttranslational modifications for studying their function and turnover in vitro and in cells.Item Light-activation of DNA-methyltransferases(2021-04-07) Wolffgramm, Jan; Buchmuller, Benjamin; Palei, Shubhendu; Muñoz-López, Álvaro; Kanne, Julian; Janning, Petra; Schweiger, Michal R.; Summerer, Daniel5-Methylcytosine (5mC), the central epigenetic mark of mammalian DNA, plays fundamental roles in chromatin regulation. 5mC is written onto genomes by DNA methyltransferases (DNMT), and perturbation of this process is an early event in carcinogenesis. However, studying 5mC functions is limited by the inability to control individual DNMTs with spatiotemporal resolution in vivo. We report light-control of DNMT catalysis by genetically encoding a photocaged cysteine as a catalytic residue. This enables translation of inactive DNMTs, their rapid activation by light-decaging, and subsequent monitoring of de novo DNA methylation. We provide insights into how cancer-related DNMT mutations alter de novo methylation in vivo, and demonstrate local and tuneable cytosine methylation by light-controlled DNMTs fused to a programmable transcription activator-like effector domain targeting pericentromeric satellite-3 DNA. We further study early events of transcriptome alterations upon DNMT-catalyzed cytosine methylation. Our study sets a basis to dissect the order and kinetics of diverse chromatin-associated events triggered by normal and aberrant DNA methylation.Item Light-activation of DNA-methyltransferases(2021) Wolffgramm, Jan; Summerer, Daniel; Mutschler, HannesThe most prominent DNA modification in mammalian cells is the 5-methylation of cytosine (5mC) in a CpG context and several severe diseases like cancer are connected to aberrant cytosine methylation. 5mC is a dynamic key epigenetic modification regulating chromatin states and therefore gene transcription, and it is highly important to get new insights into the regulation and effects of such epigenetic mark. It is necessary to be able to write 5mC at user-defined genomic loci at specific time points to analyze locus specific and time-resolved downstream effects. 5mC is written by DNA methyltransferases (DNMTs) and several approaches were made to control 5mC levels either on a global or targeted gene level. However, these approaches had low spatial and/or temporal control and resulted in off-target effects. For now, it was not possible to control the catalytic activity of DNMTs itself. This work reports the direct light-control of in vivo DNMT activity to overcome drawbacks of previous approaches. A photocaged cysteine is genetically encoded on DNMT3a to replace the Cys710 which is essential for the catalytic activity. This leads to the expression of DNMT3a proteins in an inactive state and simultaneous activation of them is achieved by light irradiation. This results in the cleavage of the caging group and the remaining of an unmodified and active DNMT3a. Since upstream processes like transfection and protein expression are uncoupled from the actual catalytic activity after light-activation, the kinetics of 5mC writing alone can be monitored. This tool is used to study the effects of several DNMT3a mutations connected to acute myeloid leukemia on the catalytic activity, providing new in vivo observations to dissect the role of these mutations. In addition, it is a great advantage to be able to write 5mC at user-defined genomic loci to specifically alter the chromatin state or to monitor protein interactions associated with 5mC at such loci. Here, locus-specificity is reached by fusing a recombinant DNMT protein with a programmable transcription activator like effector protein. Thereby, inactive DNMTs are recruited to the target locus and activated at a given time point which prevents off-target methylation. It is noteworthy that the final 5mC level is tunable by adjusting the light irradiation time. Also, time-resolved effects of DNA methylation on the transcriptome are reported, providing data on the fast consequence of DNA methylation on gene expression. In conclusion, the ability to control the activity of DNMTs with least structural change by just incorporating a single non-canonical amino acid and the rapid activation of such by light gives previous unreachable spatio-temporal control over DNA methylation in living cells. With this tool, the kinetics of downstream effects of 5mC can be monitored and new insights into the 5mC-related epigenetic network can be obtainedItem Deciphering strand-asymmetrically modified CpG dyads in the DNA double-helix(2021) Buchmuller, Benjamin Christopher; Summerer, Daniel; Linser, RasmusAlle Lebewesen müssen dafür Sorge tragen, in ihrem umfangreichen Erbgut die gerade für sie überlebensnotwendigen Gene von denen zu unterscheiden, die nicht gebraucht werden. Insbesondere mehrzellige Organismen müssen diesen Prozess zellspezifisch koordinieren, trotz dessen, dass hier dieselbe Erbinformation in allen Zellen des Individuums vorliegt. Ein Mechanismus, welcher diesem Zwecke dient, ist die Modifikation von DNA-Nukleobasen, den Bausteinen des Trägers der Erbinformation. In Säugetieren wie Mensch und Maus kommt hierbei der Methylierung der DNA-Nukleobase Cytosin am Kohlenstoffatom C5 des Pyrimidinrings eine besondere Rolle zu. Sie findet auf beiden Strängen der DNA-Doppelhelix innerhalb des kurzen Sequenzpalindroms CpG statt und trägt entscheidend dazu bei, dass hier ortsspezifisch andere molekulare Interaktionen für die Expression der Erbinformationen notwendig werden. Da das Produkt 5-Methylcytosin für weitere enzymatische Modifikationen wie der Oxidation zu 5-Hydroxymethylcytosin, 5-Formylcytosin oder 5-Carboxycytosin zur Verfügung steht, können unterschiedliche Kombinationen dieser Cytosinderivate mit gänzlich einzigartigen chemischen Eigenschaften an den komplementären CpG-Paaren im DNA-Doppelstrang vorliegen. Ein Aspekt, der unter dem Gesichtspunkt der epigenetischen Funktion dieser Derivate in Ermangelung technologischer Innovation sie in natürlichem Chromatin zu untersuchen, bislang kaum erschlossen werden konnte. Inwiefern es nun möglich ist, solche Strang-symmetrischen oder Strang-asymmetrischen Kombinationen von Cytosinderivaten in diesen CpG-Paaren auf molekularer Ebene in der DNA-Doppelhelix zu erkennen und somit gegebenenfalls zu entschlüsseln, ist Gegenstand der vorliegenden Arbeit. Ausgehend von verschiedenen Homologen einer Proteindomäne, welche symmetrisch methylierte CpG-Paare erkennen, den Methyl-CpG-bindenden Domänen (MBD), wurden aufgrund struktureller Erwägungen und funktionaler Studien der MBD–DNA-Binding, degenerierte Proteinvariantenbibliotheken erstellt. Mithilfe eines hierfür eigens entwickelten Hochdurchsatzverfahrens gelang es, Varianten zu identifizieren, die nahezu selektiv eine aus fünfzehn Paarungen obiger Cytosinderivate im DNA-Doppelstrang erkennen. Neben allgemeinen Substitutionsprofilen für verschiedene Paarungen wurden im Speziellen mehrere MBD-Varianten entdeckt, die eine neue, natürlicherweise nicht vorhandene Selektivität für 5-Hydroxymethyl- und 5-Carboxymethylcytosin-haltige CpG-Paarungen aufwiesen. Aus der weiteren biochemischen und strukturellen Charakterisierung der Bindespezifität konnten einige Erkenntnisse über die molekulare Erkennung Strang-asymmetrisch modifizierter CpG-Paarungen gewonnen werden, welche in Zukunft als Schlüssel dienen können, die epigentische Funktion der Cytosinmodifizierung im humanen Genom mithilfe solcher speziell auf sie zugeschnittenen Sonden zu entschlüsseln.Item Click-mediated enrichment of specific genomic loci(2020) Witte, Anna; Summerer, Daniel; Rauh, DanielIn all organisms, the genetic information of cells is stored in the nucleotide sequence of deoxyribonucleic acid (DNA). The human organism consists of more than 200 different somatic cell types with the same genetic information (genotype). Even though, they drastically differ in their morphology and function (phenotype), which is related to different gene expression levels. Gene expression is controlled by macromolecular interactions and epigenetic modifications on chromatin that are highly locus-specific and drive functional aspects of each locus. Even though, the compositions of macromolecules and modifications on many chromosome loci remain poorly understood, in part due to the lack of locus-specific chromatin purification methods that would allow for targeted, discovery-oriented analyses. In this work, the first enrichment method based on bio-orthogonal conjugation (“click-chemistry) with encoded programmable DNA binding domains (transcription-activator like effectors – TALEs) for purification of user-defined genomic loci was established. This click-mediated enrichment provides complementary potential compared to the existing enzymatic biotinylation strategies used in chromatin enrichment methods in the view of site-specificity and proteome-wide background. This method will enable correlations of local chromatin states with phenotypes as the key to a deeper understanding of the regulation landscape of the eukaryotic genome. As a first outlook experiment, we extended our approach to fusion constructs of specific TALE proteins and ten-eleven translocation (TET) dioxygenases for epigenetic editing in vivo. TETs catalyze the oxidation of 5-methylcytosine (5mC) to the oxidized derivates 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). In combination with the click-mediated enrichment and proteomics analysis, this will enable studying how local epigenetic changes modulate the local chromatin landscape in vivo as basis for alterations in gene expression.Item Directed evolution of lysine deacetylases(2020) Spinck, Martin; Summerer, Daniel; Musacchio, AndreaEvery cell in the human body contains the exact same genetic information in their nucleus but despite this cell function, structure and content can differs significantly. The reason for this is found in the epigenetic information, a complex code of chemical modification on the chromatin proteins and DNA which determines cellular identity. This code recently grew in complexity with the discovery of lysine acylation on histone proteins. Lysine acylation is related to lysine acetylation and spans a wide range of acyl-Coa derived modifcations like lysine propionylation, butyrylation, or crotonylation up to long chained myristoylation. All these modifications are installed and removed by a relatively small set of substrate promiscuous lysine acetyltransferases and deacetylases, respectively. The large extend of substrate promiscuity has hindered our understanding of acylation as part of the epigenetic code so far. Here I present a directed evolution-based approach to alter deacylation selectivity of lysine deacetylases allowing for the manipulate of cellular acylation patterns and I developed novel methodology to rapidly measure deacylation activity. Both methods are based on genetic code expansion, a method to genetically encode unnatural amino acids in place of an amber stop codon into proteins, including various lysine acylations. I found that by acylation of the catalytic lysine of Orotidine 5'-phosphate decarboxylase (Ura3) its activity becomes dependent on deacylation and allows for selection of E.coli cells. Through the use of acylated firefly luciferase a highly sensitive KDAC deacylation assay was established. The Ura3 selection system was able to select the E.coli Sirtuin CobB, and the human Sirtuins 1, 2, 3, 6 and 7 as well as the human histone deacetylase HDAC8. Selection of 40 million CobB mutants produced a wide range of acyl-selective mutants, as well as mutants exhibiting deallylation and boc- deprotection activity. Particularly interesting was the CobBac2 mutant, which lost all decrotonylation activity but maintained all other deacylation activities. The crystal structure of CobBac2 revealed that crotonyl binding stabilized a novel conformation of the cofactor binding loop. Addition of NAD+ caused the reaction to stop at intermediate 3, which could not be hydrolyzed due to the positioning of the cofactor binding loop. Expression of CobBac2 in mammalian cells removed all acylation except crotonylation, showing its potential to alter the epigenetic code. The same selection procedure was used to identify acetyl selective Sirt1 mutants and to isolate Sirt6 mutants with increased deacetylation activity. The acylated firefly luciferase was further developed into an assay for drug discovery. In cooperation with the COMAS we identified novel scaffolds for Sirt1 inhibition, which is comparable in potency to Ex527 but more selective for Sirt1. Screening of activators could not identify potent chemical activators, but surprisingly we found that the native C-terminal lamin A peptide strongly activates Sirt1 decrotonylation. In the future acyl-selective deacetylases will make an important contribution to a better the understanding of acylation in various biological processes such as aging, cell differentiation and metabolism.Item Chemoselective conjugation strategies for the programmable detection of epigenetic cytosine 5-modifications with transcription activator-like effectors(2019) Gieß, Mario David; Summerer, Daniel; Rauh, DanielMethylation at the cytosine (C) carbon-5 position in DNA is a reversible regulatory element of transcription in mammalian cells involved in development and disease. An active demethylation pathway through iterative oxidation of 5-methylcytosine (5mC) has been identified that leads to abasic sites at which unmodified C are restored via the base excision repair (BER) pathway. This process yields 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as partially stable intermediates. An increasing amount of data indicate that these 5-modified cytosines can also act as epigenetic regulatory elements and are involved in developmental and pathological processes. However, the function of epigenetic 5-modified cytosines are not fully understood and require sensitive typing and profiling approaches with high resolution. Engineered transcription activator-like effector (TALE) proteins have been established as programmable DNA-binders for the detection of epigenetic 5-modified cytosines. However, previous efforts to engineer new TALE selectivities did either not provide full selectivity for epigenetic 5-modified cytosines or had low affinity to target DNA. To overcome these hurdles, two novel TALE-based strategies based on chemical conjugation reactions were developed. In the first approach, a size-reduced TALE repeat with universal binding to the four canonical nucleobases and four epigenetic 5-modified cytosines was engineered. Chemoselective conjugation of 5hmC, 5fC or 5caC to dedicated blocking groups was found to abolish TALE binding. This enabled complete decoding of the three oxidized 5mC derivatives (ox5mCs) at single positions within target DNA. In the second approach, the non-canonical amino acid (ncAA) para-acetylphenylalanine (pAcF) was incorporated into dedicated positions of the universal TALE repeat. TALEs bearing pAcF maintained universal binding to cytosine 5-modified nucleobases. Furthermore, pAcF-bearing TALE repeats and 5fC-bearing target DNA could be crosslinked through oxime condensation via bifunctional hydroxylamine linkers. This enabled robust and selective enrichment of 5fC-bearing target DNA from genomic DNA (gDNA) backgrounds. Taken together, the addition of selective conjugation chemistries to TALE-based detection methods expand the toolbox for the selective, programmable analysis of epigenetic 5-modified cytosines.Item Epigenetic chromatin modification by amber suppression technology(2018-08) Neumann, Heinz; Neumann-Staubitz, Petra; Witte, Anna; Summerer, DanielThe genetic incorporation of unnatural amino acids (UAAs) into proteins by amber suppression technology provides unique avenues to study protein structure, function and interactions both in vitro and in living cells and organisms. This approach has been particularly useful for studying mechanisms of epigenetic chromatin regulation, since these extensively involve dynamic changes in structure, complex formation and posttranslational modifications that are difficult to access by traditional approaches. Here, we review recent achievements in this field, emphasizing UAAs that help to unravel protein-protein interactions in cells by photo-crosslinking or that allow the biosynthesis of proteins with defined posttranslational modifications for studying their function and turnover in vitro and in cells.Item Programmable Protein-DNA Crosslinking for the Direct Capture and Quantification of 5-Formylcytosine(2019-06-04) Gieß, Mario; Munoz-Lopez, Alvaro; Buchmuller, Benjamini; Kubik, Grzegorz; Summerer, Daniel5-Formylcytosine (5fC) is an epigenetic nucleobase of mammalian genomes that occurs as intermediate of active DNA demethylation. 5fC uniquely interacts and reacts with key nuclear proteins, indicating functions in genome regulation. Transcription-activator-like effectors (TALEs) are repeat-based DNA binding proteins that can serve as probes for the direct, programmable recognition and analysis of epigenetic nucleobases. However, no TALE repeats for the selective recognition of 5fC are available, and the typically low genomic levels of 5fC represent a particular sensitivity challenge. We here advance TALE-based nucleobase targeting from recognition to covalent cross-linking. We report TALE repeats bearing the ketone-amino acid p-acetylphenylalanine (pAcF) that universally bind all mammalian cytosine nucleobases, but selectively form diaminooxy-linker-mediated dioxime cross-links to 5fC. We identify repeat-linker combinations enabling single CpG resolution, and demonstrate the direct quantification of 5fC levels in a human genome background by covalent enrichment. This strategy provides a new avenue to expand the application scope of programmable probes with selectivity beyond A, G, T and C for epigenetic studies.Item Expanding the genetic code for site-directed spin-labeling(2019) Braun, Theresa; Drescher, Malte; Summerer, DanielSite-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy enables studies of the structure, dynamics, and interactions of proteins in the noncrystalline state. The scope and analytical value of SDSL–EPR experiments crucially depends on the employed labeling strategy, with key aspects being labeling chemoselectivity and biocompatibility, as well as stability and spectroscopic properties of the resulting label. The use of genetically encoded noncanonical amino acids (ncAA) is an emerging strategy for SDSL that holds great promise for providing excellent chemoselectivity and potential for experiments in complex biological environments such as living cells. We here give a focused overview of recent advancements in this field and discuss their potentials and challenges for advancing SDSL–EPR studies.Item Overcoming conservation in TALE-DNA interactions: a minimal repeat scaffold enables selective rerognition of an oxidized 5-methylcytosine(2018) Maurer, Sara; Buchmuller, Benjamin; Ehrt, Christiane; Jasper, Julia; Koch, Oliver; Summerer, DanielTranscription-activator-like effectors (TALEs) are repeat-based proteins featuring programmable DNA binding. The repulsion of TALE repeats by 5-methylcytosine (5mC) and its oxidized forms makes TALEs potential probes for their programmable analysis. However, this potential has been limited by the inability to engineer repeats capable of actual, fully selective binding of an (oxidized) 5mC: the extremely conserved and simple nucleobase recognition mode of TALE repeats and their extensive involvement in inter-repeat interactions that stabilize the TALE fold represent major engineering hurdles. We evaluated libraries of alternative, strongly truncated repeat scaffolds and discovered a repeat that selectively recognizes 5-carboxylcytosine (5caC), enabling construction of the first programmable receptors for an oxidized 5mC. In computational studies, this unusual scaffold executes a dual function via a critical arginine that provides inter-repeat stabilization and selectively interacts with the 5caC carboxyl group via a salt-bridge. These findings argue for an unexpected adaptability of TALE repeats and provide a new impulse for the design of programmable probes for nucleobases beyond A, G, T and C.